Polysomy 21 (extra copies of chr.21) is the most common acquired aneuploidy in B cell acute lymphoblastic leukemia (B-ALL), and constitutional trisomy 21 (Down syndrome, DS) confers a 20-fold increased risk of B- ALL. The causative molecular mechanisms of this association remain poorly understood. In preliminary studies, we identified abnormal differentiation and enhanced leukemogenesis in B cells from Ts1Rhr mice, which are trisomic for 31 genes present in the Down Syndrome Critical Region (DSCR) on human chr.21. These represent the first lymphoid-specific phenotypes reported in mouse models of trisomy 21. We also defined a B cell expression signature associated with DSCR triplication that is enriched in gene targets of the polycomb repressor complex 2 (PRC2) and highly associated with human DS-ALL. PRC2 is a histone H3 lysine 27 methyltransferase and a frequent target of mutation in cancer. Finally, an RNA-interference screen of the triplicated DSCR genes showed that Ts1Rhr B cells are selectively sensitive to knockdown of Hmgn1, which encodes a nucleosome binding protein known to promote chromatin relaxation and alter histone H3 modifications. We hypothesize that trisomy 21 promotes B-ALL through HMGN1-mediated alterations in PRC2 target gene expression. To further define the mechanistic links between polysomy 21, HMGN1, and B-ALL, and to define novel therapeutic targets in leukemias with polysomy 21, we propose the following Specific Aims: (1) Define the contribution of histone H3 modifications to B cell transformation induced by DSCR triplication. We will define the epigenetic landscape of B cells and B-ALL blasts with polysomy 21, and assess whether genetic and chemical modulation of histone H3K27 marks abrogates transformation associated with DSCR triplication. (2) Determine the effects of Hmgn1 triplication on initiation and persistence of polysomy 21 B-ALL. We will genetically modulate HMGN1 expression and assess effects on gene expression, histone modifications, and leukemogenesis in murine and human B cells with polysomy 21. There are no targeted therapies currently available for patients with polysomy 21 B-ALL. Thus, these studies address an unmet therapeutic need. In addition, this project will develop focused approaches for studying and therapeutically targeting the biologic consequences of cancer-associated copy number alterations. The applicant Dr. Andrew Lane has outlined a five-year career development plan to meet his goal of becoming an independent investigator in translational cancer biology. Dr. Lane has assembled an Advisory Committee of internationally recognized experts to provide scientific and career mentorship. He enlisted collaborators who are experts in cancer epigenetics to provide experimental advice and specific training in the field. Dana-Farber Cancer Institute is the ideal environment for completion of his scientific and career goals, given its outstanding research community and substantial record for training independent physician-scientists.